microbiology module 3

Dark Field Microscope

Dark field microscopes can be used to greatly increase the contrast between a specimen and background, resulting in a dark background with bright objects in it (Figure 3.4). Unlike brightfield or phase contrast microscopy where light passes directly through the sample, dark field microscopy reflects light off of the specimen at an angle. This reflective approach does not permit the visualization of intracellular structures. Figure 3.4 Dark Field Microscopy. The spirochete borrelia (the causative agent of Lyme disease) is often imaged using dark field microscopy. Unlike bright field or phase contrast, the background is dark, while the sample appears illuminated.

How is the total magnification of an object calculated?

Total magnification is calculated by multiplying the power of the objective and the power of the eyepiece. For instance, a 40x objective with a 10x eyepiece would make an object appear (40 x 10) 400 times larger (400x).

What is the primary purpose of a wet mount?

Wet mounts are most often performed to visualize live cells as well as the motility and behavior of an organism

Define the measurements micrometer and nanometer.

A micrometer (µm) is defined as being one-millionth of a meter and is commonly designated at 10-6 meters. A nanometer (nm) equals 10-9 m or one-billionth of a meter.

Negative staining is an alternative to the positively charged simple staining dyes listed above. A negative stain can be thought of the inverse of a simple stain. Instead of staining the microorganism, you stain everything except the microorganism. By applying the dark stain nigrosin (or India ink) to a sample, its negative charge is repelled by the negatively charged membrane, resulting in a sharp contrast between the unstained specimen and the now dark background. Note: A negative stain is only mildly invasive and may not kill the microorganism. As such, a negative stain is contraindicated for pathogenic samples.

Acid-fast staining (also known as the Ziehl-Neelsen stain) is a differential stain used to identify bacterial stains showing a high degree of resistance to decolorization. Such microorganisms are often inconclusive when using the Gram stain. Mycobacterium tuberculosis is the most common use for an acid-fast stain as it is the causative agent of tuberculosis (Figure 3.9). The staining procedure uses the red dye carbolfuchsin, initially staining all cells red. However, following the decolorization wash step, only cells with a thick lipid-based (acid-fast) protective membrane remain red. Samples are then counterstained with methylene blue to stain non-acid-fast bacterium. This staining procedure is vital in the diagnosis of tuberculosis and is most often performed on sputum samples obtained from patients. Figure 3.9 Acid-fast stain. A representative image of Mycobacterium tuberculosis is shown. The causative agent of TB is shown in red, while non-acid fast cells only retain the counterstain and are shown in blue.

The acid-fast stain is most often used to identify what specific microorganism?

Acid-fast stains are used to identify bacterial stains showing a high degree of resistance to decolorization. Mycobacterium tuberculosis is the most common use for an acid-fast stain.

Sample Detection: Staining

As most cells are relatively transparent, there are a wide variety of techniques used to prepare specimens for examination by microscopy. Stains can be used to examine tissues (muscle or connective), specific types of cells (blood, bacterial, etc.) or even organelles within an individual cell (nucleus, ER, etc.). For the purposes of this microbiology course, we will focus on both prokaryotic and eukaryotic microorganisms, their identifying traits, and the associated clinical implications. The strategies below will build upon the information regarding cell composition described in Module 1, metabolic and enzymatic properties from Module 2, and will be visually demonstrated in Lab 3.

Bright Field Microscope

Brightfield microscopes are the simplest form of light, or optical, microscopes. Light, most often emitted from a standard halogen bulb, enters the microscope from the base (bottom) and is reflected via mirrors towards the sample. As described in Lab 2, before the light reaches the sample, it first passes through a condenser converging the light beams into a focused area on the sample (Figure 3.1). The iris diaphragm controls the amount of light that passes through the sample and into the objective lens. The objective lens is the lens closest to the sample and yields the greatest amount of magnification. (Note: The degree of magnification is directly proportional to the amount of light needed. Thus, to image samples clearly at higher magnifications, more light is required). Once light passes through the sample and the objective lens, it is directed through the ocular lens, or eyepiece, to your eye. The most common power of an ocular lens is 10x. For a microscope using two lenses (objective and ocular) the total magnification of a specimen is multiplicative. Thus, a 40x objective and a 10x ocular result in a total magnification of 400x. Figure 3.1 Knowing the Microscope. The primary components of a compound microscope, often used for simple bright field microscopy are labeled accordingly.

Confocal (Laser Scanning) Microscope

Confocal (laser scanning) microscopes combine the usefulness of fluorescence microscopy with the ability to visualize cells in 3-D. Unlike light or fluorescence microscopy where light is focused a single plane (2-D), confocal microscopy can capture images in either 2-D or 3-D. Rather than using halogen (brightfield) or UV (fluorescence) light, confocal microscopes use lasers to focus on a single plane within an object and with a higher degree of accuracy (Figure 3.6). Rendering a three-dimensional image is a sequential process whereby once an image is taken, the laser then moves to an adjacent plane, captures an image, then repeats this process until the desired depth of the sample has been covered. Computer programs process the stack of 2-D images acquired, digitally combines them, and renders a 3-D reconstruction of the sample. (Practically, you can think of each 2-D plane as a sheet of paper. Each subsequent image would be like stacking another sheet upon the original and then adding another, then another, etc. Thus, as you increase the number of sheets of paper you begin to form a three-dimensional object). Figure 3.6 Confocal Microscopy. Fluorescent staining of adherent cells counterstained against the nucleus (blue), actin (green), and mitochondria (red). Notice the increased resolution and contrast compared to standard fluorescence microscopy. Individual actin filaments are clearly visible (right inset; green).

What is the primary difference between TEM and SEM?

During transmission electron microscopy the electron passes through the sample whereas during scanning electron microscopy the electron is reflected off the sample creating a three dimensional 'shell' model of the specimen.

Electron Microscope (TEM and SEM)

Electron microscope (TEM and SEM) is used to visualize incredibly small specimens. As light microscopy (brightfield, phase contrast, fluorescence, and confocal) is limited to a resolution of about 0.2 µM, it cannot efficiently visualize viruses or even some subcellular compartments. To circumvent this restriction, electron microscopes use beams of electrons (rather than light), which have significantly shorter wavelengths than light, to increases its resolution capacity to less than 1nm—that's 200x better! However, EM is labor intensive, requires samples to be fixed (killed), and the process may alter the cell structure.

In order to visualize cells, samples are most often stained with specific combinations of dyes that are taken up by the cell. Staining is often required due to the limitation of resolution on unstained cells because the flat and transparent regions of a cell may appear invisible under bright field conditions (Figure 3.2). By staining the cell with various dyes, these regions can become labeled and thus visualized. However, staining typically requires fixing the cells by heat or chemical methods before adding the dye. The cell fixation process produces its own challenges as it kills and may even distort the sample. The challenges and benefits of various staining procedures will be covered in the next section.

Figure 3.2 Brightfield Imaging. Adherent cells are imaged using a bright field microscope. As the cells are unstained, they largely appear invisible. Upon closer examination you can discern the nucleus (dark areas) as well as the plasma membrane (thin lines) outlining the shape of the cell.

Gram-negative bacteria have a relatively thin peptidoglycan layer followed by an outer membrane composed of lipopolysaccharides (LPS). This distinguishing characteristic sets Gram-negative apart from Gram-positive bacteria. During the Gram staining procedure, Gram-negative bacteria will initially retain the crystal violet dye. However, by next washing the cells with alcohol, a step referred to as the decolorization wash, the LPS and thin peptidoglycan layers of Gram-negative bacteria are unable to retain the dye, and the outer membrane is depleted of its color. Importantly, Gram-positive bacteria remain unaffected by this decolorization step. To visualize the now unstained bacteria, a secondary (counterstain) dye called safranin is added. By counterstaining with the positively charged Safranin dye, Gram-negative bacteria now retain a pink color.

Figure 3.8 Gram Staining. (A) Gram-positive bacillus, (B) Gram-negative bacillus, and (C) a mixed culture of Gram-positive coccus and Gram-negative bacillus are shown.

What is one limitation of fixing your sample?

Fixation requires you to irreversibly kill your sample. Thus, determining the motility (cell movement) of a sample is impossible. Fixation also runs the risk of distorting the specimen shape and arrangement.

Fluorescence Microscope

Fluorescence microscopes take advantage of fluorescent molecules called fluorophores to visualize cells on a dark background. Unlike brightfield, the energy of the incoming light is in the form of the ultraviolet (UV) spectrum. UV light excites different fluorophores at varying wavelengths, enabling scientists to use a wide array of colors during imaging. For instance, the green, yellow, and red fluorescent proteins (GFP, YFP, and RFP, respectively) have become important tools in microscopy (Figure 3.5). These fluorescent proteins alone can be expressed in a cell: nonspecifically illuminating the cell as a whole linked (coupled) to a normal cellular protein of interest whereby the fluorescent color is indicative of protein movement and localization or used as tags on molecules or antibodies used to designate the presence (fluorescence detected) or absence (no fluorescence) of a specific protein target. Figure 3.5 Fluorescence. Adherent cells expressing GFP (A) and mRFP (B) allow researchers to monitor the subcellular localization of specific proteins. Protein expression levels, protein localization, and trafficking can then be studied globally, as shown in the merged image (C).

Gram staining is based on what basic principle?

Gram staining, developed by Hans Christian Gram in 1884, began with the basic observation that different types of bacteria react differently to various dyes. Some bacteria readily take up a specific dye while others do not.

Gram staining was first developed by Hans Christian Gram in 1884 and is still used to this day. It began with the observations that different types of bacteria react differently to various dyes. This differentiation, based on color, divided bacteria into two categories: Gram-positive or Gram-negative (Figure 3.8). Almost a century and a half later, Gram staining remains an essential technique in microbiology laboratories.

Gram-positive bacteria have a thick cell wall with many overlapping strands of peptidoglycan. As previously discussed, a thick peptidoglycan layer provides a vital protective barrier to the surrounding environment. However, this layer is not impermeable, meaning select molecules (water, nutrients, etc.) can still pass through to the intracellular space. The Gram stain exploits this characteristic by using the dye combinations of crystal violet and iodine. Crystal violet is retained by the thick peptidoglycan cell wall and forms a stable complex with iodine (upon its addition) effectively trapping the dyes in the cell. The resulting mixture is a purple coloration of the cell. Thus, Gram-positive cells appear purple.

What is a key determinant in a bacteria being Gram-positive?

Gram-positive bacteria have a thick peptidoglycan layer. The Gram stain exploits this characteristic by using the dye combinations of Crystal violet and Iodine. Crystal violet is retained by the thick peptidoglycan cell wall and forms a stable complex with iodine (upon its addition) effectively trapping the dyes in the cell. The resulting mixture is a purple coloration of the cell.

What is the purpose of heat fixing a sample?

Heat fixing ensures the samples tightly adhere to the glass slide prior to staining (and washing) procedures.

Giemsa is also a differential stain often used in clinical settings. Combined with Wright's stain (stains blood cells), the resulting combinatorial stain can be applied to blood smears to determine the presence (or absence) of pathogenic bacteria—human (blood) cells appear purple, and bacterial cells appear as pink. This staining is most often used for the diagnosis of malaria as well as other blood parasites.

In summary, there are numerous strategies for the visualization and identification of microbes, each with its own purpose, advantages, limitations, etc. When approaching a microbe, it is often beneficial to start with the basic questions and progress into more complex diagnostic procedures. As a reminder, the above information will be visually demonstrated in Lab 3.

Microscopy

Now that we have covered the basic cellular components of both prokaryotic and eukaryotic cells (see Module 1) and how they function (see Module 2), the basic question remains: How do we visualize and detect these differences? Dating back as far as the late 1600s, glass lenses have been used to enlarge (or magnify) objects of interest. Thus, the microscope, and the field of microscopy, was born. The microscope is perhaps the most important resource for studying biology at the microscopic level. To better appreciate how a microscope reveals the microbial world we first must address the units of measurement commonly used by researchers. The micrometer (µm) is one-millionth of a meter and is commonly designated at 10-6 m, while the nanometer (nm) equals 10-9 m or one-billionth of a meter. For perspective, the unaided eye can resolve (see clearly) objects typically > 100 µm. However, cellular components, organelles, etc. can be as small as 0.2 µm in diameter.

As light passes through a microscope, what is the last piece that light passes before reaching your eyes?

Once light passes through the sample and the objective lens it is directed through the ocular lens, or eyepiece, directly into your eye.

As the Gram stain distinguishes between bacteria with a thick peptidoglycan wall and those without (or very thin), this is referred to as a differential stain. Differential staining is a generalized term used for any staining technique that separates specimens into further subgroups. This process most often utilizes at least two dyes.

One of the disadvantages of the Gram stain is the requirement of the cells to be fixed (attached) to a glass slide. Tightly adherent cells are required to prevent sample loss during the staining and wash steps. The most common method to fix a sample is via heat fixation. By this process, samples are added to a glass slide and then passed through a flame until all liquid in the sample has been removed. Alternatively, chemical fixation strategies are also available and include the use of paraformaldehyde, ethanol, or methanol. Although these processes fix the sample to the slide, it also kills the microorganism. As such, characteristics related to motility (movement) are not possible.

Phase-contrast microscopy provided what benefits to imaging?

Phase contrast microscope can provide detailed images of live cells without staining. By using specialized condensers and objectives, a phase contrast microscope amplifies the slight differences between cells and the surrounding medium (background) to make the cells highly distinguishable.

Phase Contrast Microscope

Phase contrast microscopes have a distinct advantage over bright field microscopy in that they are often able to visualize certain structures that would otherwise be invisible (Figure 3.3). Thus, a phase contrast microscope can provide detailed images of live cells without staining. By using specialized condensers and objectives, a phase contrast microscope amplifies the slight differences between cells and the surrounding medium (background) to make the cells highly distinguishable. For these reasons, phase contrast microscopy can be used to visualize cell movements (motility), such as swimming or gliding, without altering the cell morphology commonly brought about from treating the cells with a fixing agent. Note: Although phase contrast microscopy can image live cells in great detail, often scientists will still use organelle specific dyes to highlight a particular region of the cell. For instance, although the cell membrane, nucleus, and other organelles are readily apparent, a researcher interested in the trans-Golgi (TGN) region may still add a dye that specifically binds within the TGN just to make it more prominent in the final acquired image. Figure 3.3 Phase Contrast. Noticeable detail and depth is achieved to what would normally be a nearly invisible cell. Note the defined cell periphery (plasma membrane) and internal organelles.

What are the two critical factors that influence your ability to see an object?

Resolution and contrast. Resolution refers to the distance between two objects at which the objects still can be seen as separate. Poor or low resolution means two (or more) objects may appear as one. The contrast is the difference in light absorbance between two objects. Poor contrast gives a high background and makes the visualization of multiple objects difficult. For instance, trying to identify 2 dark colored objects at night (low light = low contrast) versus the same 2 objects in the middle of a sunny afternoon (bright light against 2 dark objects = high contrast).

Scanning electron microscopes (SEM)

Scanning electron microscopes (SEM) also use a beam of electrons, but the image is obtained as the electrons reflect off (not through) the surface of the specimen. Samples are coated with either gold or palladium to enhance electron reflection. Thus, SEM can only be used to generate a detailed three-dimensional shell model of the surface of a specimen, as shown in Figure 3.7B. As with TEM, live samples cannot be viewed using SEM. Figure 3.7 Electron Microscopes. (A) Image from a TEM of a nanoparticle shows the 2-D rendering, while a (B) SEM image is able to capture 3-D architecture of the same nanoparticle.

Scanning transmission electron holography microscopes (STEHM)

Scanning transmission electron holography microscopes (STEHM) also use an electron beam but coupled with a holography technique to study surfaces of proteins and subcellular structures. It has the capacity to magnify subatomic structures up to 20 million times larger than what can be viewed with the naked eye. While EM can resolve 1 nm (10-9), STEHM has the capacity to resolve 35 pm (a picometer is one-trillionth of a meter, or 10-12) and possibly even smaller.

Wet mount is a basic form of sample preparation for viewing live samples. A small liquid culture (usually just a drop) containing a microorganism of interest is prepared, added to a slide, and then covered with a glass coverslip. The coverslip is present to both protect the objective and the specimen while also holding the microorganism in place. Note: Heat fixing is not performed. Wet mounts are used to observe the motility and behavior of an organism.

Simple staining uses a solution of a positively charged dye, such as methylene blue, crystal violet, safranin, or fuchsin, to bind to and stain the negatively charged membrane of the microorganism. This simple technique is often used to quickly observe the size, shape, and arrangement of cells.

Unlike brightfield microscope, fluorescence microscopes illuminate samples through what spectrum?

The energy of the incoming light is in the form of the ultraviolet (UV) spectrum.

If you wish to increase the amount of light going into a microscope, what part would you adjust?

The iris diaphragm controls the amount of light that passes through the sample and into the objective lens. Thus, as you open the iris more light is permitted to pass through to illuminate the sample.

Two critical factors influence our ability to see an object: the resolution and the contrast. The resolution refers to the distance between two objects at which the objects still can be seen as separate. Thus, the closer two objects are to each other, the greater the resolution requirement will be to maintain viewing the two objects as separate. The contrast is the difference in light absorbance between two areas (objects). The lower the contrast between an object and its background, the harder it will be to see that object. Thus, the greater the contrast between two areas the easier it will be to visualize.

The type of microscope largely influences the power of resolution and the degree of contrast. The varying types used in microbiology are outlined briefly below.

Transmission electron microscopes (TEM)

Transmission electron microscopes (TEM) use thin slices of a sample, heavily treated and coated in preservatives, and placed between the electron beam source and the detector. An image is formed from the interactions of the electrons as they pass through the thin sectioning of the sample. This process can be used to visualize subcellular organelles, substructures, and viral particles as shown in Figure 3.7A.

What is the distinguishing feature of dark field microscopy?

Unlike bright field or phase contrast microscopy where light passes directly through the sample, dark field microscopy reflects light off of the specimen at an angle. The resulting image is an exceptionally dark background and a vibrant specimen.